1) Field of the Invention
The present invention relates to water soluble particles comprising a biological macromolecule and a method of isolating a biological macromolecule from an aqueous solution with simultaneous dehydration of the protein, to provide protein biological macromolecule particles. The present invention also relates to water miscible organic solvents comprising the protein precipitated therein. The present invention may find particular application in preparing enzymes for use as biocatalysts; preparation of therapeutic proteins for use in pharmaceutical formulations; production of cleansing agents comprising enzymes; production of paints, varnishes, coatings, films and the like comprising proteins which impart protective and/or antifouling properties; production of films, polymers, inks, coatings, electrodes and/or optical materials comprising proteins for diagnostic kits and/or biosensor applications; use of proteins for studies of molecular recognition, molecular binding and inhibitor binding in non-aqueous media; and preparation of protein based food additives. Additionally the precipitated biological macromolecule may thereafter be dissolved in organic solvents for use in at least some of the aforementioned applications as well as in solid phase chemistry such as in the preparation of catalysts for attachment, cleavage and/or modification of compounds bound to an insoluble support.
2) Description of the Related Art
Proteins are used in a great variety of applications. However, generally speaking, for therapeutic purposes it is necessary to have a preparation of protein substantially free from impurities for use. There are many ways purification may be achieved such as by differential centrifugation, selective precipitation, solvent extraction and chromatographic processes. Additionally it is often desirable to dehydrate or dry the protein prior to use, that is remove water from the protein, in order to facilitate handling and/or improve shelf life.
Typically proteins may be dehydrated by freeze drying, vacuum drying or air drying techniques commonly known in the art. However these techniques suffer from a number of disadvantages. For example, the drying processes are not generally very quick and can be extremely expensive. Moreover, even freeze-drying may lead, particularly in the case of enzymes and fragile proteins, to a decrease in protein function. In order to preserve protein function additional stabilising excipients are often added. However, addition of stabilising excipients may in itself be undesirable particularly, for example, from a regulatory point of view for proteins to be used therapeutically.
U.S. Pat. No. 5,198,353 discloses a method of preparing a stabilised enzyme dispersion. There is described a method of coprecipitating a polymer and an enzyme from an aqueous solution in order to produce a finely dispersed enzyme for use in aqueous based liquid detergents. The polymer and enzyme are precipitated by the addition of either salts or organic solvents. When using an organic solvent as the precipitant it is disclosed that the organic solvent is added to the aqueous protein/polymer solution slowly with vigorous stirring in order to precipitate the protein. However the method and the amount of organic solvent added is such that there is not extensive and rapid dehydration of the protein.
U.S. Pat. No. 5,589,167 and U.S. Pat. No. 5,753,219 disclose excipient stabilisation of polypeptides treated with organic solvents. Polyols such as trehalose are disclosed as stabilising dry or aqueous polypeptides treated with organic solvents. However, there is no suggestion that the polyols could be used to coprecipitate with the protein on addition to an organic solvent or the relevance/importance of dehydrating the protein.
Randen et al (J. Pharm. Pharmacol., 1988, 40, 761–766) describes the coprecipitation of enzymes with water soluble starch as an alternative to freeze-drying. Starch of molecular weights 12 700 and 100 000 is disclosed as a coprecipitant of krill proteases when mixed with an organic solvent of acetone, ethanol or isopropanol. The particles produced after precipitation are described as irregular needles with low density with a size in the range of 200–700 μm. After drying the particles had to be further processed by milling or grinding to obtain a more uniform size distribution.
In a later paper citing the Randen et al paper, Bustos et al (J. Chem. Tech. Biotechnol., 1996, 65, 193–199) describe the use of additional polymeric compounds for use as coprecipitants. The polymeric compounds disclosed are hydrolysed collagen, casein and maltodextrins PSM 10 (12,100 Mw) and PSM 100 (100,000 Mw).